Disorders of extracellular inorganic phosphate (Pi) concentration and impairment in Pi reabsorption are common clinical problems in the general population. Aging, diabetes mellitus, malignancy, alcoholism, post-organ transplantation, AIDS, and several therapeutic drugs are well known to cause or to be associated with hypophosphatemia or hyperphosphatemia, mainly by affecting renal tubular Pi transport. The kidney plays a critical role in the regulation of Pi homeostasis. Pi is freely filtered across the glomerulus and most of the Pi is reabsorbed along the proximal tubule via a sodium gradient-dependent process (Na/Pi cotransport). The Na/Pi cotransporter is located on the apical brush border membrane (BBM) of the proximal tubule. The evidence to date indicates that regulation of the overall renal tubular Pi transport by dietary, hormonal, or metabolic factors occurs at the level of the proximal tubular BBM Na/Pi cotransport system. The specific aims of this proposal are to determine: 1) the role of lipid microdomains/ lipid rafts, alterations in the diffusion and/or clustering of Na/Pi proteins in the regulation of Na/Pi cotransport activity, 2) the role of clathrin, caveolae and dynamin in trafficking; specifically, internalization of Na/Pi protein from the apical membrane, 3) the role of SNAREs in trafficking, especially targeting of Na/Pi protein to the apical membrane and 4) the effect of direct alterations in cholesterol content on these processes of lateral and vertical diffusion of Na/Pi Protein. The models utilized will consist of Pi-adaptation and modification of cholesterol content, in Oppossum Kidney Cells (OK), a cell culture line. The methods employed will range from well-known biochemical approaches of transport rate, protein measurements and biotinylation experiments to newer biophysical techniques of imaging and co-localization such as Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) and Confocal Immunofluorescence Microscopy.